1. Field of the Invention
The invention relates to novel eukaryotic neurocan proteins, subunits thereof and nucleic acid encoding therefor, which are useful for providing soluble, biologically active heterologous proteins in hosts. The invention is further related to hosts transformed with this nucleic acid and methods for producing soluble heterologous proteins in hosts using such molecules.
2. Description of the Background Art
A large body of data implicates a family of receptors, the selectins (lectin-EGF-complement binding-cell adhesion molecules (LEC-CAMs)), in the initial interactions between leukocytes and vascular endothelia leading to lymphocyte homing, platelet binding, and neutrophil extravasation (Hallman et al., Biochem. Biophys. Res. Comm. 174:236-243 (1991); Lawrence and Springer, Cell 65:859-873 (1991); Luscinskas et al., J. Immunol. 142:2257-2263 (1989); Watson et al., Nature, 349:164-167 (1991); Watson et al., J. Cell Biol., 115:235-243 (1991). L-selectin is involved in lymphocyte homing to peripheral lymph nodes. P-selectin participates in adhesion of activated platelets. E-selectin seems to facilitate T-cell infiltration at sites of cutaneous inflammation (Picker et al., Nature, 349:796-799 (1991); Shimizu et al., Nature, 349:799-802 (1991)). All three may be involved in neutrophil extravasation at sites of tissue damage or infection (Stoolman, Cell, 907-910 (1989). The cell surface expression of these three receptors is differentially regulated, and binding of one receptor may have significant effects on the expression of other selectins and on integrin adhesion receptors (Kuijpers et al., J. Immunol., 147:1369-1376 (1991); Lo et al., J. Exp. Med., 173:1493-1500 (1991); Spertini et al., Nature 349:691-694 (1991)) .
The three known selectins, L-Selectin (leukocyte adhesion molecule-1 [LECAM-1], LAM-1, gp90MEL), E-Selectin (LECAM-2, endothelial-leukocyte adhesion molecule-1 [ELAM-1]), and P-Selectin (LECAM-3, GMP-140), each contains a domain with homology to calcium-dependent lectins (C-lectins), and EGF-like domain, and several complement binding protein-like (CBP) domains (Bevilacqua et al., Science, 243: 1160-1165 (1989); Johnston et al., Cell, 56:1033-1044 (1989); Lasky et al., Cell, 65:1045-1055 (1989); Tedder et al., J. Exp. Med., 170:123-133 (1989)) . Identification of the C-lectin domains has led to an intense effort to define carbohydrate ligands for these glycoproteins. There is now general agreement that E-Selectin recognizes NeuNAc .alpha.2-3 Gal .beta.1-4 (Fuc .alpha.1-3) GlcNAc (sialyl-Lewis.sup..chi., or sLe.sup..chi.) and related oligosaccharides (Berg et al., J. Biol. Chem., 265:14869-14872 (1991); Berg et al., J. Cell Biol. 114:343-349 (1991); Lowe et al., Cell, 63:475-484 (1990); Phillips et al., Science, 250:1130-1132 (1990); Tiemeyer et al., Proc. Nat'l Acad. Sci. U.S.A., 88:1138-142 (1991); Tyrrell et al., Proc. Nat'l Acad. Sci. U.S.A., 88:10372-10376 (1991); Walz et al., Science, 250:1132-1135 (1990)). P-Selectin has been reported to recognize the Lewis.sup..chi. structure (Gal .beta.1-4 (Fuc .alpha.1-3) GlcNAc) (Larsen et al., Cell, 63:467-474 (1990)) and/or sLe.sup..chi. (Polley et al., Proc. Nat'l Acad. Sci. U.S.A., 88:6224-6228 (1991)); although other ligands are possible (Moore et al., J. Cell. Biol., 112:491-499 (1991)) . See Foxall et al., J. Cell. Biol., 117:895-902 (May, 1992).
The recruitment of leukocytes from the blood is one of the most dramatic cellular responses to tissue damage and inflammation, and is central to the physiologic trafficking of lymphocytes. Leukocyte extravasation is exquisitely regulated in vivo by mechanisms of selective leukocyte-endothelial cell (EC) recognition, which can display extraordinary specificity in relation to the inflammatory stimulus, the stage of the inflammatory response, and the tissue site or organ involved. Examples include the almost exclusive attachment of eosinophils to venules in allergic reactions, the specific recruitment of neutrophils early in acute inflammation, and the tissue-selective interaction of lymphocyte subsets with high endothelial venules (HEVs) in organized lymphoid tissues.
Adhesion receptors (ARs) mediate and help direct leukocyte-EC interactions (Table I; reviewed in Springer, Nature 346:425-433 (1990); Pober and Cotran, Transplantation 50:537-544 (1990); Berg et al., Vascular Adhesion Molecules, Cellular and Molecular Mechanisms of Inflammation, Vol. 2, pp. 111-129 (1991)). Paradoxically, however, individual receptors often participate in multiple leukocyte-EC interactions that are quite independently regulated in vivo. For example, the vascular E-selectin (ELAM-1) binds both neutrophils and skin-homing memory T-cells, yet is thought to support selective recruitment of neutrophils during acute inflammation and of cutaneous memory T-cells during chronic inflammation in the skin (Picker et al., Nature 349:796-799 (1991), and references cited therein). Such observations cannot be explained by simple lock-and-key models of cell-cell recognition; and seem to require a more complex control of leukocyte-EC interactions in vivo.
A general model in which leukocyte-EC recognition is viewed as an active process requiring at least three sequential events. First, interaction is initiated by binding of constitutively functional leukocyte ARs to EC counterreceptors. In the best characterized examples, such primary adhesion is mediated by lectin-carbohydrate interactions involving leukocyte or vascular "selectins" and their cognate oligosaccharide ligands (see Table I). This initial adhesion is transient and reversible, unless followed by a second event (activation of the leukocyte by specific chemoattractant or cell contact) mediated signals capable of triggering secondary ARs whose function is activation dependent. Interaction of the activation-dependent AR with its EC counterreceptor, the third step, results in strong, sustained attachments completing the process of recognition. The best characterized activation-dependent ARs are heterodimeric integrins of the .beta.2 (CD18) or .beta.1 (CD29) classes. See Butcher, Cell, 67:1033-1036 (Dec., 1991).
TABLE I ______________________________________ Leukocyte Endothelium ______________________________________ Step 1. Primary Adhesion Pathways Lectin-Carbohydrate L-selection (L, N, M) Lymph node addressin CLA (smTL) E-selectin (ELAM-1) sialyl Lewis .chi. (N, M) E-selectin sialyl Lewis .chi. (N, M) P-selectin (GMP140, CD62) Other ? Mucosal addressin Step 2. Chemoattractant/Activating Factors Intercrine Family Interleukin-8 (N, L) hMGSA/GRO.alpha. (N) Platelet factor 4 (N, M) RANTES (mTL, M) HuMIP-1.alpha. (CD8.sup.+ TL, BL) HuMIP-1.beta. (vTL, ?M) I-309 (M) Monocyte chemoattractant (M) protein-1 Others Lipids Platelet activating factor (N) Leukotriene B4 (N, M) Others Other Chemoattractants C5a (N) Formyl peptides (N, M) Interleukin-2 (sTL) Cell Contact-Mediated E-selectin binding (N) CD44 (L) Step 3. Activation-Dependent Adhesion Pathways Integrins LFA-1 (.alpha.L.beta.2) (L &gt; N, M) ICAM-1, ICAM-2 Mac-1 (.alpha.M.beta.2) (N, M, sL) ICAM-1, others .rho.150, 95 (.alpha..chi..beta.2) (N, M, sL) ? VLA-4 (.alpha.4.beta.1) (M, mL &gt; vL) VCAM-1 ______________________________________
See Butcher et al., Cell, 67:1033-1036 (Dec., 1991), which is entirely incorporated herein by reference.
A number of recent studies indicate that chondroitin sulfate proteoglycans are involved in modulating cell interactions in developing nervous tissue (Hoffman et al., 1988; Crossin et al., 1989; Perris and Johansson, 1990; Margolis et al., 1991; Perris et al., 1991; Snow et al., 1991; Brittis et al., 1992) and are components of astroglial axon barriers (Snow et al., 1990a,b; McKeon et al., 1991; Oakley and Tosney, 1991). (The references cited herein by author and year are listed in the references section presented herein before the claims section.)
These findings indicate that extracellular chondroitin sulfate proteoglycans may act as repulsive molecules which modulate cell-cell and cell-matrix interactions by providing a mechanism for diminishing adhesive forces, thereby permitting cell rounding, division, differentiation, and cell movement in developing brain, and indicate the need for information on their primary structures, which will permit more detailed functional studies including work aimed at exploring the roles of specific protein domains.
Accordingly, there is a need to provide biologically active polypeptides having activities related to cell adhesion, leukocyte-endothelial cell recognition and differentiation and development of nervous tissue.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.